Method of use of a platelet/nucleated cell concentrate from bone marrow or blood to treat a bone defect

ABSTRACT

The present invention is directed to methods and compositions regarding the preparation of an cell concentrate, such as, for example, an osteogenic cell concentrate, from a physiological solution, such as bone marrow aspirate, blood, or a mixture thereof. In specific embodiments, the invention provides methods and compositions utilizing two physiological solution-processing techniques, particularly in a point of care environment, wherein centrifugation is not employed.

The present application claims priority to U.S. patent application Ser.No. 10/811,549, filed Mar. 29, 2004, which claims priority to U.S.Provisional Patent Application Ser. No. 60/458,354 and to U.S.Provisional Patent Application Ser. No. 60/528,583, filed Dec. 10, 2003,all of which applications are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The present invention generally regards the fields of bone therapy, bonemarrow processing, and medicine, such as orthopedic medicine. In aspecific embodiment, the present invention is directed to the processingof physiological product, such as bone marrow aspirate, through adual-step filter system either step of which lacks centrifugation.

BACKGROUND OF THE INVENTION

Bone graft substitutes (BGS) are commonly used as a less-invasivealternative to autograft for the repair of osseous defects. Autograft,however, remains the gold standard in grafting applications due to itssupply of a structural scaffold, osteoinductive growth factors, andosteogenic cells. A promising surgical option to autograft is the use ofa composite graft consisting of a BGS combined with bioactive (i.e,osteoinductive and osteogenic) elements. Examples of bioactive elementsinclude platelets (which secrete osteoinductive growth factors) and stemcells (which differentiate into osteogenic bone-forming cells). Sourcesof these bioactive elements include bone marrow aspirate and blood,which are less invasive to procure from a patient than autograft. Withthis in mind, techniques have been developed to procure and concentratebioactive elements from bone marrow aspirate and blood for use in tissuehealing applications. Centrifugation is typically used to separateosteogenic/osteoinductive cells from other blood constituents based ondifferences in density. However, preparation of cell-rich suspensionscan be arduous in addition to requiring the purchase and maintenance ofa centrifuge in the operating room. Accordingly, a point-of-caresurgical technique that could concentrate stem cells and platelets fromblood or bone marrow aspirate without the need for centrifugation isdesirable. This would give a surgeon a convenient method to rapidlyprepare a bioactive BGS that would be a viable and less invasivealternative to autograft.

In Connolly et al. (1989), marrow extracted from rabbits wasconcentrated by simple centrifugation, isopyknic centrifugation, andgravity sedimentation, all of which increased the nucleated cell counton average compared to whole marrow. The osteogenic effect of bonemarrow was tested in rabbits, using a chamber that had been implanted ina peritoneal cavity (ectopic site) and in a delayed-union model(orthotopic site). Osteogenesis was accelerated in both sites afterconcentration of marrow elements after centrifugation, but not aftergravity sedimentation. Although a method to increase the nucleated celldensity within bone marrow aspirate was described, centrifugation wasutilized to concentrate stem cells in marrow. Furthermore, theconcentrate was not used in conjunction with an osteoconductive scaffoldmaterial.

PCT Patent Application WO 96/27397 provides a plasma-buffy coatconcentrate that comprises plasma, platelets, and fibrinogen. When theconcentrate is combined with a fibrinogen activator in sufficientconcentration to initiate clot formation, a wound sealant is formed.Also provided is a method for processing blood to produce theplasma-buffy coat concentrate. The method comprises centrifuginganticoagulated blood to remove red blood cells and to produce aplasma-buffy coat mixture. Water is removed from the mixture byhemofiltration to produce the plasma-buffy coat concentrate. Afibrinogen activator is mixed with the plasma-buffy coat concentrate toproduce a wound sealant that can be used for multiple clinicalindications, including bone-grafting applications.

U.S. Pat. Nos. 6,010,627 and 6,342,157 provide a device and a method forconcentrating a blood fraction, typically plasma, to provide aconcentration of blood procoagulant proteins, such as fibrinogen, andcellular components, such as platelets, white blood cells, or buffy coatcells. Water is removed from plasma by hemofiltration to produce aplasma-buffy coat concentrate. The resultant concentrate is suitable foruse in the preparation of coagulum-based wound sealants. The methodutilizes a hemofilter, which is a type of hollow fiber filter used forblood processing, in order to concentrate cells and proteins within ablood fraction. The prior art defines an ultrafiltration unit having asemi-permeable membrane with a molecular weight cut-off of about 30,000daltons, which allows for the concentration of fibrinogen protein,useful for coagulation upon addition of a fibrinogen activator to thecell/protein concentrate. However, U.S. Pat. Nos. 6,010,627 and6,342,157 do not describe the fiber filter for the concentration ofmesenchymal stem cells and platelets from either whole blood or bonemarrow aspirate. Accordingly, a larger cut-off filter (i.e., exceeding30,000 daltons) may be used in the present invention to concentratecells since the recovery of proteins is not critical. Because largercut-off filters have higher flow rates and require lower operatingpressure than smaller cut-off filters, the filters in the presentinvention will require less time and force to operate compared to thefilters disclosed therein.

Muschler et al. (2002; U.S. Pat. Nos. 5,824,084 and 6,049,026) provide amethod for preparing a composite BGS in which bone marrow aspirate ispassed through a porous substrate. The osteoprogenitor cells areselectively retained in the substrate, resulting in a composite graftthat contains an enriched (i.e., greater) number of progenitor cellscompared to an equivalent volume of bone marrow aspirate. A method isprovided for passing marrow through an implantable porous scaffold thatacts as an affinity column for mesenchymal stem cells. The stem cellsare selectively retained in the substrate, resulting in a compositegraft that contains an enriched (i.e., greater) number of stem cellscompared to an equivalent volume of bone marrow aspirate. However, thedisclosure of Muschler does not describe methods and compositions forthe enrichment of platelets.

PCT Patent Application WO 00/61256 and U.S. Pat. No. 6,398,972 describean automated method and apparatus for producing platelet-rich plasma ora platelet concentrate from a physiological solution, preferably blood.Processing is carried out by a centrifuge that receives a disposablecontainer having two chambers. Whole blood is placed into one of the twochambers, and the centrifuge is then operated to cause the red bloodcells to sediment to the bottom of one chamber, resulting in asupernatant of platelet-rich plasma. The centrifugation is stopped,which causes the platelet-rich plasma to drain to the second chamber.The platelet-rich plasma in the second chamber is then centrifuged asecond time by restarting the centrifuge. The centrifuge is stopped,resulting in: (1) red blood cells in one chamber; (2) plateletconcentrate at the bottom of the second chamber; and (3) platelet poorplasma as the supernatant in the second chamber. A portion of theplatelet poor plasma is removed from the second chamber, leaving aremaining portion of the platelet poor plasma and platelet concentratein the second chamber. The remaining platelet concentrate is suspendedin the platelet-poor plasma remaining in the second chamber to obtainplatelet rich plasma.

Thus, the present invention as described herein provides a need in theart of clinical therapy, particularly bone therapy, lacking in thepresent methods.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a system, method, and compositionsthat are beneficial for use in the repair of, for example, osseousdefects. More particularly, the present invention provides a method toprepare a cell concentrate from a physiological solution (preferablybone marrow aspirate, blood, or a mixture thereof) at the point of careand, optionally, without the need for centrifugation. Although the cellconcentrate is preferably utilized for osteogenic treatments, inalternative embodiments the cell concentrate is analyzed, such as fordiagnosis, drug testing, or prognosis purposes, for example.

In a particular embodiment of the invention, there is a method ofpreparing a cell concentrate, comprising the steps of providing aphysiological solution not previously subjected to centrifugation;subjecting said physiological solution to a filter to produce a filterretentate and a permeate solution, wherein said filter retentatecomprises platelets, nucleated cells, or both per unit volume greaterthan in the physiological solution and wherein said permeate solutioncomprises plasma and red blood cells; and removing the filter retentatefrom the filter. In a specific aspect, the filter is a leukocytereduction filter.

In specific embodiments, the providing step may be further defined ascombining an additional solution with the physiological solution,wherein the additional solution comprises water or a hypotonic solution,such as sodium chloride.

In another embodiment of the present invention, there is a method ofpreparing a cell concentrate, comprising the steps of providing aphysiological solution; subjecting the physiological solution to a firstfiltration device to isolate nucleated cells, platelets, or both fromthe physiological solution, the isolating step producing a firstproduct; and subjecting said first product to a second filtration deviceto produce a second product, the second product comprising a number ofnucleated cells, platelets, or both per unit volume greater than in thephysiological solution. In a specific embodiment, the first filtrationdevice is a nucleated cell filtration device, which may be furtherdefined as a leukocyte reduction filtration device, and in anotherspecific embodiment, the second filtration device is further defined asa hollow fiber filtration device.

A filtration device, such as, for example, the second filtration device,may comprise a pore size between about 0.05 μm and about 5 μm or, morepreferably between about 0.2 μm and about 0.5 μm.

Methods of the invention lack a centrifugation step, in preferredembodiments.

The physiological solution comprises bone marrow aspirate, blood, or amixture thereof, in specific embodiments, and in some embodiments thenucleated cells comprise stem cells, connective tissue progenitor cells,osteoprogenitor cells, chondroprogenitor cells, or a mixture thereof.The stem cells are mesenchymal stem cells, hematopoietic stem cells, orboth, in some embodiments. In other embodiments the method furthercomprises the step of delivering the second product to a bone defect inan individual and/or comprises the step of admixing a scaffold materialto said second product to produce a scaffold material/second productmixture. In some embodiments, the method may also comprise the step ofdelivering the scaffold material/second product mixture to a bone defectin an individual.

The scaffold material may be comprised of a block, paste, dust, cement,powder, granule, putty, liquid, gel, solid, or a mixture thereof and/orit may be comprised of a ceramic, a polymer, a metal, allograft bone,autograft bone, demineralized bone matrix, or a mixture thereof. Thescaffold material may be biodegradable and is preferablyosteoconductive, osteoinductive, or osteogenic. In a specificembodiment, the scaffold material is comprised of synthetic material,natural material, or a combination thereof.

In a specific embodiment, the method further comprises the step ofadmixing a biological agent with the second product, the scaffoldmaterial, or a combination thereof, and the biological agent admixedwith the scaffold material may be further defined as the biologicalagent being comprised on the scaffold material, in the scaffoldmaterial, or both. In particular embodiments, the scaffold materialitself is the biological agent.

In another embodiment of the present invention, there is a cellconcentrate generated by methods described herein.

In an additional embodiment of the present invention, there is a methodof increasing nucleated cell concentration and/or platelet concentrationfrom a physiological solution, comprising the steps of providing aphysiological solution; subjecting the physiological solution to anucleated cell filtration device to isolate nucleated cells, platelets,or both from the physiological solution, the isolating step producing afirst product; and subjecting the first product to a fibrous filtrationdevice to produce a second product, the second product comprising anumber of nucleated cells, platelets, or both per unit volume greaterthan in the physiological solution.

The providing the bone marrow aspirate step may be further defined asaspirating the bone marrow from an individual into a first syringe toproduce a bone marrow aspirate. Preferably, the first syringe comprisesan anti-coagulant, an isotonic solution, or both.

In a further specific embodiment, the subjecting the bone marrowaspirate to a nucleated cell filtration device is further defined asintroducing the bone marrow aspirate to a first housing device, saidfirst housing device connected in-line to a leukocyte reduction filterand said leukocyte reduction filter connected in-line to a secondhousing device, wherein the leukocyte filter permits passage of plasmaand red blood cells through said filter but inhibits passage ofnucleated cells, platelets, or both; introducing a purge solution to thefilter to produce a purge solution/nucleated cell/platelet mixture; andretrieving the purge solution/nucleated cell/platelet mixture from saidfilter.

The subjecting the first product to a filtration device may comprisesubjecting the first product to a hollow fiber filtration device and/orthe subjecting the first product to a filtration device may comprisesubjecting the first product to the filtration device, the devicecomprising a filter, wherein the feed direction of the first productthrough the filtration device is nonparallel to the flow of the firstproduct across the membrane.

In a specific embodiment, the providing the blood step may be furtherdefined as aspirating the blood from an individual into a first syringeto produce a blood aspirate, for example. The first syringe may comprisean anti-coagulant, an isotonic solution, or both. In a specificembodiment, the subjecting the blood aspirate to a nucleated cellfiltration device is further defined as introducing the blood aspirateto a first housing device, said first housing device connected in-lineto a leukocyte reduction filter and said leukocyte reduction filterconnected in-line to a second housing device, wherein the leukocytefilter permits passage of plasma and red blood cells through said filterbut inhibits passage of nucleated cells, platelets, or both; introducinga purge solution to the filter to produce a purge solution/nucleatedcell/platelet mixture; and retrieving the purge solution/nucleatedcell/platelet mixture from said filter.

The providing the bone marrow aspirate/blood mixture step may be furtherdefined as aspirating the bone marrow and blood from an individual intoa first syringe to produce a bone marrow aspirate/blood aspirate. Thefirst syringe may comprise an anti-coagulant, an isotonic solution, orboth. The subjecting the bone marrow aspirate/blood aspirate to anucleated cell filtration device may be further defined as introducingthe bone marrow aspirate/blood aspirate to a first housing device, saidfirst housing device connected in-line to a leukocyte reduction filterand said leukocyte reduction filter connected in-line to a secondhousing device, wherein the leukocyte filter permits passage of plasmaand red blood cells through said filter but inhibits passage ofnucleated cells, platelets, or both; introducing a purge solution to thefilter to produce a purge solution/nucleated cell/platelet mixture; andretrieving the purge solution/nucleated cell/platelet mixture from saidfilter.

In an additional embodiment of the present invention, there is a methodof treating a bone defect in an individual, comprising the steps ofobtaining a physiological solution comprising nucleated cells,platelets, or both; subjecting the physiological solution to a nucleatedcell-filtration device to isolate nucleated cells, platelets, or both,said isolating step producing a first product; subjecting the firstproduct to a hollow fiber filtration device to produce a second product,the second product comprising a number of nucleated cells, platelets, orboth per unit volume greater than in the physiological solution; anddelivering the second product to the bone defect in the individual. Themethod lacks a centrifugation step, in specific embodiments, although inalternative embodiments a centrifugation step is utilized, such as, forexample, to remove adipocytes and non-cellular fatty matter from theplatelets and nucleated cells.

The bone defect may comprise a break, fracture, void, diseased bone,loss of bone, brittle bone, weak bone, bone injury, or bonedegeneration. The method may further comprise the step of admixing ascaffold material with the second product. The physiological solution isblood, bone marrow aspirate, or a mixture thereof, in some embodiments.

The physiological solution is preferably obtained from the individual.In a specific embodiment, the method occurs at a point-of-care in ahospital facility or a health care provider facility. In anotherspecific embodiment, the method further comprises administering to theindividual an additional bone defect therapy. The additional bone defecttherapy comprises fracture repair, surgery, bone excision, implantdelivery, external stimulation, or a combination thereof. In specificembodiments, the nucleated cells comprise leukocytes, stem cells,connective tissue progenitor cells, osteoprogenitor cells,chondroprogenitor cells, or a mixture thereof. The stem cells may bemesenchymal stem cells, hematopoietic stem cells, or a mixture thereof.The delivering the second product to the individual may compriseapplying the second concentrated product directly to the bone defect. Ina specific embodiment, the applying is with a scoop, scoopula, syringe,rod, or spatula.

In another specific embodiment, the method further comprises the step ofadmixing a biological agent with the second product, the scaffoldmaterial, or a combination thereof. The biological agent admixed withthe scaffold material may be further defined as the biological agentbeing comprised on the scaffold material, in the scaffold material, orboth.

In an additional embodiment of the present invention, there is a methodof preparing an osteogenic cell concentrate from a physiologicalsolution comprising subjecting the physiological solution to at leastone filtration step, wherein the method preferably lacks centrifugation.

In another embodiment, there is a kit for preparing a cell concentrate,comprising a first filtration device and a second filtration device,both of which are housed in a suitable container. In a specificembodiment, the first filtration device and second filtration device arehoused in a suitable container. In another specific embodiment, thefirst filtration device is a nucleated cell filtration device and/or thesecond filtration device is a microfiltration device. Themicrofiltration device may be further defined as a hollow fiberfiltration device.

The kit may further comprise a scaffold material housed in a suitablecontainer and/or the kit may further comprise a biological agent housedin a suitable container.

In another embodiment of the present invention, there is a kit fortreating a bone defect, comprising a plurality of cells housed in asuitable container, wherein said cells are nucleated cells, platelets,or both. The kit further comprises a scaffold material housed in asuitable container, in specific embodiments. The kit may furthercomprise a biological agent housed in a suitable container.

In another embodiment of the present invention, there is a kitcomprising an apparatus for the preparation of a cell concentrateobtained by a method described herein.

In a particular embodiment, there is a method of preparing a cellconcentrate, comprising the steps of providing a physiological solutionnot previously subjected to centrifugation; subjecting the physiologicalsolution to a leukocyte reduction filter to produce a filter retentateand a permeate solution, wherein the filter retentate comprisesplatelets, nucleated cells, or both per unit volume greater than in thephysiological

solution and wherein the permeate solution comprises plasma and redblood cells; and removing the filter retentate from the filter.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings, in which:

FIG. 1 provides an exemplary schematic showing the filtration of marrrowaspirate through a leukocyte reduction filter to selectively isolatenucleated cells (including mesenchymal stem cells) and/or platelets froma physiological solution.

FIG. 2 provides a schematic showing an exemplary hand-held operation ofa hollow fiber filtration device.

FIG. 3 illustrates a schematic showing the filtration of isolatednucleated cells and/or platelets through a hollow fiber filter toincrease the concentration of the cells.

FIGS. 4A-4D provide an exemplary schematic showing operation of firstfilter (leukoreduction-type filter) to selectively recover osteogeniccells (i.e., platelets and nucleated cells) from a physiologicalsolution.

FIG. 5 illustrates an exemplary schematic showing operation of a secondfilter (hollow fiber filter) to concentrate osteogenic cells (i.e.,platelets and nucleated cells) recovered from first filter. The syringecontaining the recovered osteogenic cells is connected to thehollow-fiber filter, which is operated in cross-flow mode. In cross-flowfiltration, the feed stream is recirculated between a feed syringe andretentate syringe tangentially to the membrane, establishing a pressuredifferential across the membrane. This causes some of the particles topass through the membrane. Remaining particles continue to flow acrossthe membrane. Using an appropriate membrane pore size (0.2 to 0.5 μm),recovery solution, but not cells, are able to pass through the filtermembrane of the hollow fiber filter.

FIG. 6 provides an exemplary schematic showing a concentrate osteogeniccell suspension resulting from operation of second filter. Cross-flowfiltration fractionates a portion of the recovery solution from thesuspended cells, thereby increasing the concentration of the cells inthe retentate syringe.

FIG. 7 is an exemplary schematic showing operation of second filter(hollow fiber filter) to concentrate osteogenic cells (i.e., plateletsand nucleated cells) recovered from first filter. The syringe containingthe recovered osteogenic cells is connected to the hollow-fiber filter,which is operated in dead-end mode (this is accomplished by cappingretentate syringe port). Unlike cross-flow filtration, dead endfiltration does not involve the recirculation of the feed stream isrecirculated between a feed syringe and retentate syringe tangentiallyto the membrane. Pressure from the advancing feed syringe establishes apressure differential across the membrane. This causes some of theparticles to pass through the membrane. Using an appropriate membranepore size (0.2 to 0.5 μm), recovery solution, but not cells, are able topass through the filter membrane of the hollow fiber filter.

FIG. 8 is an exemplary schematic showing a concentrate osteogenic cellsuspension resulting from operation of second filter in dead end mode.Dead end filtration fractionates a portion of the recovery solution fromthe suspended cells, thereby increasing the concentration of the cellsremaining in the feed syringe.

FIGS. 9A-9E provide an exemplary schematic showing operation of fatreduction filter to decrease fat particle content in physiologicalsolution and a leukofilter to selectively recover osteogenic cells(i.e., platelets and nucleated cells) from said physiological solution.

FIGS. 10A-10E illustrate a specific embodiment wherein the filtrationprocess utilizes an aggregate filter, such as a cellular and/ornon-cellular fat reduction filter.

FIG. 11 shows representative fields of view of nucleated cell counts ofblood, pre-processed BMA, and post-processed BMA.

FIG. 12 illustrates a schematic of an exemplary filter configurationused to prepare platelet rich concentrate from blood.

FIG. 13 demonstrates platelet recovery for two exemplary starting bloodvolumes.

FIG. 14 shows concentration of PDGF-AB in whole blood and recoveredplatelets.

FIG. 15 shows concentration of TGF-β1 in whole blood and recoveredplatelets.

FIG. 16 shows concentration of VEGF in whole blood and recoveredplatelets.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. Furthermore,as used herein, the terms “including”, “containing”, and “having” areopen-ended in interpretation and interchangeable with the term“comprising”.

The term “allograft bone material” as used herein is defined as bonetissue that is harvested from another individual of the same species.Allograft tissue may be used in its native state or modified to addressthe needs of a wide variety of orthopaedic procedures. The vast majorityof allograft bone tissue is derived from deceased donors. Bone is about70% mineral by weight. The remaining 30% is collagen and non collagenousproteins (including bone morphogenic proteins, BMPs). Allograft bonethat has been cleaned and prepared for grafting provides a supportmatrix to conduct bone growth, but is not able to release factors thatinduce the patient's biology to form bone cells and create new bonetissue.

In a preferred embodiment, the allograft is cleaned, sanitized, andinactivated for viral transmission.

The term “biological agent” as used herein is defined as an entity thatis added to the bone graft substitute to effect a therapeutic end, suchas facilitation of bone ingrowth, prevention of disease, administrationof pain relief chemicals, administration of drugs, and the like.Examples of biological agents include antibiotics, growth factors,fibrin, bone morphogenetic factors, angiogenic factors, bone growthagents, bone proteins, chemotherapeutics, pain killers, bisphosphonates,strontium salt, fluoride salt, magnesium salt, and sodium salt.

The term “blood” as used herein refers to circulating tissue composed ofa fluid portion (plasma) with suspended formed elements (red bloodcells, white blood cells, platelets). In a specific embodiment of theinvention, the physiological solution refers to whole blood, such as,for example, that provided by a donor, obtained from the circulatorysystem of a patient, or a mixture thereof. The blood may be obtained byany suitable means, such as in the form of a blood aspirate, forexample.

The term “bone graft substitute (BGS)” as used herein is defined as anentity for replacing bone tissue and/or filling spaces in a bone tissue.The term “bone defect” as used herein is defined as a bone defect suchas a break, fracture, void, diseased bone, loss of bone, brittle bone orweak bone, injury, disease or degeneration. Such a defect may be theresult of disease, surgical intervention, deformity or trauma. Thedegeneration may be as a result of progressive aging. Diseased bonecould be the result of bone diseases such as osteoporosis, Paget'sdisease, fibrous dysplasia, osteodystrophia, periodontal disease,osteopenia, osteopetrosis, primary hyperparathyroidism,hypophosphatasia, fibrous dysplasia, osteogenesis imperfecta, myelomabone disease and bone malignancy. The bone deficiency may be due to adisease or condition, such as a disease that indirectly adverselyaffects bone. Furthermore, the bone malignancy being treated may be of aprimary bone malignancy or may be metastatic, originating from anothertissue or part of the body.

The term “bone marrow” as used herein refers to soft, gelatinous tissuethat fills bone cavities. It is comprised of red bone marrow, whichcomprises stem cells, progenitor cells, precursor cells, and functionalblood cells, and yellow bone marrow, which mainly stores fats. Thus, redbone marrow is myeloid tissue that is actually producing blood cells.Red marrow produces red blood cells, white blood cells, and platelets. Askilled artisan is aware that children comprise much red marrowthroughout the body, but in adults it is most concentrated in the flatbones, such as the hipbone. Leukocytes (white blood cells) are alsoproduced in bone marrow and, in some embodiments, are involved in immunedefenses. In fact, marrow transplants can treat some types ofimmunodeficiency. Bone marrow is also referred to as medulla ossium. Ina specific embodiment, the bone marrow comprises bone marrow aspirate(bone marrow drawn via syringe from an individual's bone), which askilled artisan recognizes inevitably contains some peripheral blood.

The term “bone marrow aspirate” as used herein refers to the materialobtained upon aspirating bone marrow from a bone, such as by needle.

The term “nucleated cell/platelet fraction” as used herein refers to asolution derived from bone marrow, blood, or a mixture thereof thatcomprises at least nucleated cells and/or platelets. The nucleated cellfraction comprises mesenchymal stem cells, nucleated connective tissueprogenitor cells, nucleated hematopoietic cells, or endothelial cells,or a mixture thereof. In a specific embodiment, the fraction comprisesleukocytes. In a specific embodiment, the nucleated cell/plateletfraction does not substantially comprise plasma or red blood cells. Thefinal cell concentration in some embodiments will contain by volume lessthan approximately 30% of the original plasma and RBC volume. The cellfraction (which may contain some plasma and red cells) can be made tocoagulate by mixing it with an appropriate clotting initiator, such asfollowing the filtration steps and prior to application to a bonedefect. An ionic calcium solution (such as calcium chloride solution)and/or thrombin could initiate clotting to form a coagulum.

The term “centrifugation” as used herein refers to the rotation in acompartment of an apparatus, said compartment spun about an axis for thepurpose of separating materials.

The term “concentrate” as used herein refers to a composition thatcomprises a greater concentration of a specific component, for example,a particulate or particulates, compared to a parent source.

The term “cross-flow filtration” or “cross-flow mode” as used hereinrefers to tangential flow filtration, which regards the recirculation ofa retentate across the surface of the membrane filter. In specificembodiments, it refers to the direction of feed of a product through adevice being perpendicular to the direction of flow across the membrane.In some embodiments the term is defined as filtration comprisingmultiple passes over a membrane.

The term “dead-end filtration” or “dead-end mode” as used herein refersto filtration wherein the direction of feed of the product and thedirection of flow across the membrane are parallel. In some embodimentsthe term is defined as filtration comprising a single pass over amembrane. In other embodiments, dead-end filtration comprisessubstantially no tangential flow filtration.

The term “filter retentate” as used herein refers to the compositionthat is retained by a filter upon passage of a physiological solutionacross the filter. In particular embodiments, it comprises nucleatedcells, platelets, or both. In certain embodiments, the filter retentatecomprises very minor amounts of plasma and/or red blood cells. In aspecific embodiment, the volume of a filter retentate is about 1 mL orgreater.

The term “filtration” as used herein refers to the process of passing aliquid comprising particular matter through a porous material for thepurpose of separating the liquid from at least some of the particularmatter, for separating some particular matter from other particularmatter, or both.

The term “hollow fiber filtration device” as used herein is a filtrationdevice comprising a hollow tubular outer covering, wherein inside thehollow tubular outer covering there are individual tubes whose walls arefilters; the individual tubes lay in a direction parallel to the lengthof the tube. In specific embodiments, the direction of feed of a productthrough a device is perpendicular to the direction of flow across themembrane. In some embodiments it generally comprises tangential flowfiltration, which regards the recirculation of a retentate across thesurface of the membrane filter. In alternative embodiments, there is notangential flow filtration.

The term “mesenchymal stem cell” as used herein refers to pluripotentprogenitor cells located in bone marrow that can differentiate into avariety of non-hematopoietic tissues including bone, cartilage, tendon,fat, muscle, and early progenitors of neural cells.

The term “microfiltration” as used herein refers to separation ofparticles from a fluid, wherein at least one microfiltration devicecomprises at least one filter having a pore size of about 0.05 μm toabout 5 μm. In a specific embodiment of the present invention, cellsand/or cell fragments are separated from at least some fluid, whereinthe microfiltration capabilities of the filter permit passage of thefluid through the pores, thereby retaining the cells and/or cellfragments.

The term “osteoconductive” as used herein refers to the ability of amaterial, such as a scaffold material, to allow new bone ingrowth.

The term “osteogenic” as used herein refers to a material thatstimulates growth of new bone tissue.

The term “osteoinductive” as used herein refers to the ability to formbone in an ectopic (i.e., non-bony) body site.

The term “permeate solution” as used herein refers to the particles andliquid that is smaller than the pore size of a filter and thereforepasses through the filter membrane.

The term “plasma” as used herein refers to blood plasma, which is thepale yellow fluid component of whole blood comprising water, proteins,electrolytes, sugars, lipids, metabolic waste products, amino acids,hormones, and/or vitamins.

The term “purge solution” as used herein refers to a solution thatfacilitates exodus of a bone marrow or blood derived cells from afilter. In specific embodiments, the purge solution is comprised atleast in part of an isotonic saline solution or a colloidal solutionsuch as albumin or dextran, or a mixture thereof. A skilled artisanrecognizes that the purge solution may be comprised of water,electrolytes, proteins, carbohydrates, and/or gelatin, and so forth.

The term “scaffold material” as used herein refers to a material thatfacilitates bone growth upon administration to a bone defect with aosteogenic concentrate, such as is derived from a bone marrow. In aspecific embodiment, the scaffold material is comprised at least in partof a synthetic material, a natural material, or both. In furtherspecific embodiments, the scaffold material is comprised ofbiocompatible material that facilitates, permits, or enhances the layingdown of new bone matrix, bone growth and/or bone ingrowth. In additionalspecific embodiments, ceramics, such as calcium sulfate or calciumphosphate, a polymer, a metal, allograft bone, autograft bone,demineralized bone matrix, a mixture thereof, and so forth. In morespecific embodiments, the scaffold material may be a block, paste,cement, powder, granule, putty, gel, or so forth. In a specificembodiment, the scaffold material is osteoconductive, osteoinductive,osteogenic, or a combination thereof. In other embodiments, the scaffoldmaterial breaks down over time when placed in the body. In additionalspecific embodiments, the scaffold material is considered a matrix, acarrier, a solution, a solid, a gel, and the like. In specificembodiments, the scaffold matrix comprises a viscous hydrogel, such as,for example, a carboxymethylcellulose-based hydrogel. In a specificembodiment, the scaffold matrix comprises a JAX® Advanced Bone VoidFiller, and/or or JAX®-tcp (tri-calcium phosphate). In additionalspecific embodiments, the scaffold material comprises a plurality ofJAX® toy jack shaped bone graft substitutes. The scaffold material maybe a dust, powder, granule, chip, putty, tablet, mixture thereof, and soforth.

The term “second filter” as used herein refers to a filter that issubsequent to a first filter in a process comprising concentratingcells. In particular embodiments, at least one additional step may beincluded between the first and second filters, and in alternativeembodiments this at least one additional step may comprise use of afilter. Further steps to concentrate the cells may be utilized followingthe second filter.

The term “serum” as used herein refers to the aqueous component of ananimal fluid remaining after coagulation (that is, remaining after clotformation removes fibrinogen, prothrombin, and other clotting factorsfrom blood plasma).

The term “stem cell” as used herein refers to an unspecialized cell thatgives rise to a specific specialized cell.

II. The Present Invention

The present invention provides novel methods and compositions directedto the generation and/or enrichment of a cell concentrate, such as anosteogenic cell concentrate, from a physiological solution, such as bonemarrow aspirate, blood, or a mixture thereof. The procedure employs asystem wherein the physiological solution is processed through at leastone filtration step, and the system preferably lacks centrifugation.However, in alternative embodiments centrifugation may be utilized.

Thus, in some embodiments the methods of the present invention compriseonly one filtration step. In other embodiments, the methods of thepresent invention comprise two or more filtration steps. In oneembodiment of the present invention, there is a method of preparing acell concentrate by providing a physiological solution and subjecting itto a filter to produce a filter retentate and a permeate solution,wherein the filter retentate comprises platelets, nucleated cells, orboth per unit volume greater than in the physiological solution andwherein said permeate solution comprises plasma and red blood cells.Following this filtration step, the filter retentate may comprisesubstantially the majority of platelets and nucleated cells from theoriginal physiological solution, and the permeate solution may comprisesubstantially the majority of plasma and red blood cells from theoriginal physiological solution. Regarding the filter retentate, thereis at least about a four-fold, five-fold, six-fold, seven-fold, or moreincrease in concentration of platelets and/or nucleated cells over thatof the physiological solution from which they are filtered. In otherterms, about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% ofplatelets and/or nucleated cells are filtered from the physiologicalsolution using methods of the present invention. Regarding the permeatesolution, about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of plasma andred blood cells are filtered from the physiological solution, which maybe defined as thereafter being present in the permeate solutionfollowing filtration, using methods of the present invention.

In one embodiment of the present invention, a single filtration step ofa physiological solution concentrates nucleated cells, includingleukocytes, and platelets while concomitantly removing plasma and redblood cells from the physiological solution. The resultant concentratefrom this single step may be applied directly to a bone defect,processed further using a non-filtration step (such as, for example,adding a biological agent), combined with a scaffold material beforeapplication to the bone defect, or subjected to at least one morefiltration step, such as to further concentrate the cells and/orexchange the solution in which the cells are comprised.

A skilled artisan recognizes based on the disclosure provided hereinthat a use of the invention is to obtain a concentrated composition ofplatelets and nucleated cells, including leukocytes, from aphysiological solution. This is in contrast to many known processes inthe art that remove leukocytes from blood, for example, as beingundesired. The filter retentate comprising the leukocytes is the desiredcomposition, as opposed to a composition to be discarded. In otherwords, the present invention removes leukocytes from a physiologicalsolution such as blood for the purpose of utilizing them, as opposed tobeing part of a waste product. This relates to a specific embodiment ofthe invention being providing cells that release growth factors, such asleukocytes and platelets, which upon administration to a bone defectresults in cell migration to the wound, cell proliferation, anddifferentiation into bone cells. The known blood processing methods thatdiscard leukocytes are trying to reduce or inhibit an immunogenicresponse, given that the ultimate destination for the composition is forsystemic administration to an individual, such as one requiring a bloodtransfusion. In contrast, the present invention relates to localadministration to a wound, as opposed to systemic circulation, such as,for example, with autotransfusion of a cell concentrate to a bonedefect.

In other embodiments, the invention is unique in that it is the first tocombine two existing blood processing techniques (leukocyte reductionfiltration and hollow fiber filtration) into one method in order toincrease nucleated cell count (which can include mesenchymal stem cells,connective tissue progenitor cells, chondroprogenitor cells, and/orosteoprogenitor cells) and platelet concentration, optionally withoutthe need for centrifugation. Furthermore, when the concentratedphysiological solution is combined with a scaffold material, such as anosteoconductive material, the invention results in a composite BGS thatenhances the formation of new bone compared to the scaffold or cellconcentrate alone. In additional specific embodiments, the cellconcentrate is mixed with a compound, such as with a powder or liquid,to form an injectable putty, which may optionally comprise at least oneclotting initiator. Examples of clotting initiators are solutionscomprising calcium ions (e.g., calcium chloride solution) or thrombin,or both. In further specific embodiments, a biological agent is combinedwith the cell concentrate, the scaffold material (in the material, onthe material, or both), or the mixture thereof prior to or uponadministration to the bone defect.

Although there are methods in the art that employ centrifugation toconcentrate stem cells for use as an osteogenic bone marrow preparation,the current invention employs filtration to concentrate both nucleatedcells (including stem cells) and platelets, resulting in a marrowconcentrate. This filtration optionally obviates costs associated withpurchasing and maintaining a centrifuge in the surgery room, and it isalso a less arduous method for processing marrow than the centrifugationmethods used by others. Furthermore, the current invention provides amethod for concentrating both osteogenic (mesenchymal stem cells) andosteoinductive (platelets) elements of bone marrow, whereas knownmethods regard concentration of one or the other, but not both.

In comparison, the present invention disclosed herein uses filters, notimplantable scaffolds, to isolate and concentrate both stem cells andplatelets. The current invention provides a method for concentratingboth osteogenic (mesenchymal stem cells) and osteoinductive (platelets)elements of marrow, whereas, for example, Muschler (Muschler et al.,2002; U.S. Pat. Nos. 5,824,084 and 6,049,026) describes a method toconcentrate only mesenchymal stem cells from marrow. When combined withan osteoconductive scaffold, a concentrated marrow suspension of bothstem cells and platelets should result in better bone healing comparedto a marrow concentrate containing only stem cells. Furthermore, thepresent invention results in a concentrated cellular suspension that canbe delivered to a bone defect by syringe injection through a needle.This should allow for minimally invasive delivery of the cellconcentrate to a bone defect. The invention described by Muschlerrequires the use of a non-injectable osteoconductive scaffold, whichprecludes delivery of the cell concentrate/scaffold to a bone defect viainjection. In other embodiments, a minimally invasive delivery, such asvia syringe injection, is utilized.

Thus, a cell concentrate, the novel preparation of which is providedherein, is used to accelerate bone healing in a variety of bone graftingapplications. Kits are also provided.

In one step of the present invention, the physiological solution isobtained, such as from an individual to which it will subsequently bedelivered following concentration, and it is subjected to at least onefiltration step, which may be a first filtration step in a processcomprising more than one filtration steps, that selectively recovers orisolates nucleated cells, which comprise osteogenic cells, such asmesenchymal stem cells, and leukocytes; and platelets, which secreteosteoinductive growth factors, such as the exemplary PDGF, TGF-β,insulin-like growth factor-I (IGF-I), insulin-like growth factor-II(IGF-II), and vascular endothelial growth factor (VEGF), or mixturesthereof. Certain non-osteogenic components, such as red blood cells andplasma, which make up a large percentage of the sample volume, are notsubstantially recovered during the sole (or first) filtration step.

In one embodiment of the present invention, the blood or bone marrowfraction to be concentrated comprises an anticoagulant, providedpreferably at the time of withdrawal, generally using an exemplarycitrate-based anticoagulant. Any citrate-based anticoagulant issuitable, for example. Standard donor blood collection bags, forexample, contain citrate-based anticoagulants. In a particularembodiment, the anticoagulant heparin is added, such as, for example,when bone marrow aspirate is used.

In particular embodiments, the physiological solution, such as the bloodor bone marrow, comprises an additional component, such as anothersolution that acts as a diluent and/or source of hypotonicity. In theembodiment wherein the additional component is a hypotonic solution,upon introducing it to the physiological solution the salt concentrationthereby becomes lower outside the cells comprised in the physiologicalsolution than the salt concentration inside the cells. Thus, an osmoticgradient is generated wherein water flows into the cells, and, inspecific embodiments, causes them to swell such that they adhere betterto the filter. This solution, which may be referred to as Solution A inthe Examples, may be, for example, water or a hypotonic sodium chloridesolution that is less concentrated than isotonic (normal), which is, forexample, about 0.9%.

Depending on the configuration of the one or more filters, the sole orfirst filter (a leukocyte reduction filter, in some embodiments)utilizes the tendency of certain types of cells (e.g., mesenchymal stemcells) to adhere to foreign substances. When passed through the filter,nucleated cells, such as stem cells, and platelets are adsorbed on thefiber surfaces within the filter, whereas red blood cells and plasma donot adsorb, and consequently pass through the filter. A purge solution,which may also be referred to as a recovery solution, is then flushedthrough the filter to remove the entrapped cells, thereby allowingrecovery of the selected osteogenic/osteoinductive cells. These cellsmay then be analyzed and/or be applied to a wound site (such as a bonedefect), optionally combined with a scaffold material before beingapplied to a wound site, or further processed. The recovery solution mayin some embodiments be hypertonic or isotonic, such that they are equalor greater than normal saline concerning sodium chloride concentration.

In other embodiments, the recovered osteogenic/osteoinductive cells arethen subjected to a second filtration step, wherein the platelets areincreased in concentration. The filtration device of this step may be ofany kind, so long as it concentrates nucleated cells, platelets, orboth. In preferred embodiments, this step comprises subjecting thesolution obtained from the first step to a hollow filtration device,such as those known in the art. The device preferably separates cellsfrom a portion of the liquid in which they are suspended, therebyincreasing the cellular concentration. A skilled artisan recognizes thatthe process is beneficial to the individual, given that a greater amountof therapeutic material may be delivered in a significantly smallervolume. In many embodiments, without such concentration of product, sucha large volume would be prohibitive to apply to the defect. This secondstep may be performed multiple times.

In preferred embodiments, the second filtration device comprises atubular filter, as opposed to a disk filter. In additional specificembodiments, the tubular filter is housed in an outer tube comprisinghollow tubes within it and, in preferred embodiments, the flow throughthe tubular filtration device is parallel to the flow through the tubeswithin the tubular filtration device. In a specific embodiments, thepore size on the walls of the tubes is about 0.05 μm to about 5 μm, ismore preferably about 0.1 μm to about 1 μm, and is most preferably about0.2 μm to 0.5 μm.

In some embodiments of the present invention, the osteogenic cellconcentrate is combined with a scaffold material and delivered to a bonedefect. Any delivery method is appropriate as long as it maintains theintegrity of the composition and provides therapy for the bone defect.The delivery method may be via syringe, scoop, spatula, scoopula, rod,tube, and so forth. In other embodiments, a viscous setting material isadded to the osteogenic concentrate or to the osteogenicconcentrate/scaffold material composition to facilitate retention of theconcentrate or concentrate/scaffold mixture at the bone defect site. Theviscous setting material may comprise a clotting factor (such ascalcium, thrombin, or mixture thereof), bone marrow aspirate, blood,platelet rich plasma, fibrinogen/thrombin, a cement, a slurry, a paste,a combination thereof, and the like. In a specific embodiment, a cement,slurry, or paste, such as one or more comprising a calcium saltincluding calcium sulfate and/or calcium phosphate, is utilized in thepresent invention, such as for the viscous setting material.

Any of the surfaces of the filtration devices used in the presentinvention that contact a physiological solution and/or the resultingconcentrate are preferably inert to the components and are preferablynot substantially cytotoxic. In preferred embodiments, for example,where it is desirable to include cells such as nucleated cells and/orplatelets concentrate, the contact surfaces are substantiallynoncytotoxic. Suitable materials include polycarbonates, polyurethane,acrylics, ABS polymers, polysolfone, polyethersulfone, mixed celluloseester, polyester, and the like.

In embodiments wherein the sterility of the osteogenic concentrate orother composition must be maintained, as in the preparation of theconcentrate for a bone defect therapy, any filtration surface thatcontacts any of the relevant compositions and/or the concentrate ispreferably sterile or readily sterilizable. Commercially availablefiltration units can be sterilized by treating with agents such as gammairradiation, ethylene oxide, formalin, hydrogen peroxide, sodiumhypochlorite, heat, steam, and so forth. Sterile filtration units arecommercially available for hematologic uses. Syringes and other fluiddelivery systems are generally commercially available in sterile form asare various valves and stopcocks that are designed to attach to syringesand other blood processing products.

In alternative embodiments of the present invention, a bone marrowconcentrate is generated by the methods described herein. The bonemarrow concentrate is then utilized for an application other than a bonedefect, such as for use in or augmentation of a bone marrow transplant.

Alternatively, the cell concentrate comprising or consisting essentiallyof platelets and nucleated cells but not plasma or red blood cells isnot prepared at the point of care, such as being obtained as acommercially prepared composition or being prepared from an individualprior to a point of care service and administered to a bone defect ofthe same individual or another individual thereafter.

In particular embodiments of the present invention, there is a cellconcentrate prepared by a method described herein.

III. Specific Embodiments

The previous discussion was directed to the general embodiments of theprocesses described herein. The following section provides specificembodiments to the procedures, although one of skill in the art would beable to make adjustments to the following steps for optimization of amethod for concentrating a product. For example, these specificembodiments may be directed to processing from whole blood, and askilled artisan recognizes which modifications would be helpful tooptimize this method.

In a general embodiment of the present invention, a single stepfiltration process concentrates the desired cells, said cellsexemplified by nucleated cells and platelets, through their isolationfrom a starting solution, such as bone marrow, whole blood, or a mixturethereof. This filtration step thereby removes plasma and red blood cellsfrom the desired cells. The resultant filtrate comprising the desiredcells is applied to a bone defect, optionally in a composition alsocomprising a scaffold material. An exemplary schematic of thisembodiment is illustrated in FIG. 1.

In one specific embodiment, the following steps are utilized in themethod:

Step 1, Obtain bone marrow: An appropriate amount of bone marrow(approximately 5 cc or greater, such as for bone grafting applications)is obtained, such as, for example, is aspirated into a syringe filledwith an equal or lesser volume of an isotonic saline and a suitableanti-coagulant (e.g., citrate-based or heparin-based). In someembodiments, the bone marrow is obtained by another process, such ascommercial purchase. In other embodiments, whole blood is the primarysource of the concentrate, and it is processed, such as mixed with asuitable volume of anti-coagulant (e.g., citrate-based orheparin-based), prior to subjection to the nucleated cell-reductionfilter of the next step.

Step 2, Filtration of marrow aspirate through a nucleated cell (such asa leukocyte) reduction filter to selectively recover nucleated cells,such as mesenchymal stem cells, and platelets (FIG. 1): The marrowaspirate is injected into a housing device, such as the small collectionbag 10. An in-line leukocyte reduction filter 12 is placed between thecollection bag 10 and a second housing device, such as the drain bag 14.The marrow aspirate is gravity-fed through the nucleated cell reductionfilter 12 into the drain bag 14. The nucleated cell/platelet fraction(containing mesenchymal stem cells and platelets) is trapped within thefilter 12 while the remaining blood constituents pass through. A syringe18 is then used to backflush the filter with a suitable volume (>about 5ml) of a purge solution (e.g., Dextran 40 and albumin solution) to allowthe recovery of the buffy coat cells in a second sterile syringe 16.

Step 3, Filtration of cells suspended in purge solution through a hollowfiber filter to increase the concentration of the nucleated cells andplatelets: The operation of the hollow fiber filtration device is shownschematically in FIG. 2. The syringe containing the cell-laden purgesolution (labeled syringe A 20) is attached to one end of the filtrationdevice 22, for example, by a Luer lock 23 at a first port 21. A secondsyringe (labeled syringe B 24) is attached to the opposing end through asecond port 25 to collect the retenate (the cells that do not passthrough the filter 30). A third syringe (labeled syringe C 26) isconnected and is attached to a third port 27 to receive the filtrate(the liquid component, i.e., purge solution, that passes through thefilter). The sample is passed back and forth between the two opposingretenate syringes (syringes A 20 and B 24) to allow circulation of theretentate and separation of the cellular and liquid components. Theliquid component is passed through the semi-permeable filter membrane 30into syringe C 26; cells cannot pass through the semi-permeable filtermembrane 30 and are collected within the fibers 32 of the filter. In aspecific embodiment, although other means are applicable, the followingsteps are performed in order to expel the cell concentrate from thefilter 30 into a sterile syringe: (i) syringe C 26 is removed from thefiltrate port 27 and the port 27 is capped; (ii) an empty retenatesyringe (either A 20 or B 24) is removed from a retenate port (either 21or 25, respectively); (iii) a syringe filled with a purge solution(preferably isotonic saline) is connected to the said available retenateport (either 21 or 25, respectively); and (iv) the purge solution, thevolume of which is approximately equal to the priming volume of thefilter 30, is injected into the filter 30 to expel the cell concentrateinto the second retenate syringe (either A 20 or B 24, respectively).

FIG. 3 illustrates a general embodiment showing that this filtrationstep fractionates the nucleated cell/platelet fraction and purgesolution, thereby allowing the reduction of the volume of solution inwhich the cells are suspended. Again, a container 16 (which in someembodiments may be syringe A 20 or B 24 as described in FIG. 2)comprising the purge solution and buffy coat from the first filtrationstep delivers the solution to the hollow fiber filter 22, through whichthe solution is filtered. Ultimately, the purge solution will reside ina container 27, such as the illustrative syringe, and the desired cellconcentrate is delivered to a container 29, such as the illustrativesyringe, for eventual delivery to a defect. Thus, the net effect is anincrease in the number of cells per unit volume of fluid.

Step 4, Mixing of cell concentrate with scaffold material: Theconcentrated cellular suspension is injected into a container holding ascaffold material, such as a granular material. In some embodiments, avariety of scaffold materials are used. The concentrate flows into thefree space within and/or between individual scaffold granules. If thefree volume available exceeds that of the cellular concentrate, then acarrier material (wherein the carrier material acts as a handling agent,acts to increase differentiation of mesenchymal cells towards anosteoblastic lineage, or both) may be mixed with the cell concentrate toachieve a volume that is equivalent to fill the free intergranularand/or intragranular space of the scaffold material. Examples of carriermaterials include bio-fluids, such as coagulated blood, marrow,platelet-rich plasma, or a synthetic material, such as a hydrogel,powder, or granules. The cell concentrate may be combined with thecarrier material by syringe mixing or by mixing in a container, using arod, spatula, or other suitable instrument. The mixture is then injectedinto a container holding the granular scaffold material and allowed toflow into the intragranular and/or intergranular space. The cellconcentrate/scaffold mixture can be delivered to an exposed defect orcan be delivered percutaneously via syringe.

Whole blood (rather than marrow aspirate) could be processed in asimilar manner as described in the previous embodiment. The resultingconcentrate would be rich in platelets (but not mesenchymal stem cells)and could be mixed with a scaffold material as described in the previousembodiment.

A combination of bone marrow and blood could also be processed asdescribed in the first embodiment. The resulting concentrate would berich in both platelets and mesenchymal stem cells and could be mixedwith a scaffold material as described in the first embodiment.

In a specific embodiment, following delivery of a cellconcentrate/scaffold material mixture to a bone defect, the defectand/or the bone or bone tissue it is comprised in is tested. Forexample, the bone may be tested for quantity and/or quality of the bone,such as at the bone defect. The tests may comprise assaying bonedensity, strength, rate of bone formation, quality of bone, and soforth. Any suitable test may be utilized, and, in some embodiments,histology, radiographs and/or mechanical tests are employed. Thepercentage of the bone defect filled with bone following delivery of thecell concentrate/scaffold material mixture may be assayed, and, in someembodiments, it is compared to surrounding bone. In some embodiments,there is a test for increased deposition of calcium followingadministration of concentrate or concentrate/scaffold mixture.

In particular embodiments, a bone marrow aspirate is obtained forprocessing through a method described herein. Although bone marrowaspirate may be obtained by other means, a specific marrow aspirationtechnique is described: following induction of anesthesia and sterileskin preparation, a small incision (less than about 1 cm) is made alongthe posterior iliac crest. A bone marrow aspiration needle is advancedthrough this incision into the intramedullary cavity of the iliac crest.A small sample of bone marrow (less than about 4 mL) should be aspiratedinto a 10-mL syringe containing heparinized saline (about 1000units/mL). After collection of marrow, the syringe is inverted severaltimes to ensure mixing with the anticoagulant. Additional aspirationscan be taken using the same technique through separate corticalperforations spaced at least about 1 cm apart.

In particular embodiments, blood is obtained for processing through amethod described herein. Although blood may be obtained by other means,a specific blood aspiration step is described: following sterile skinpreparation, a small needle (18-21 gauge) infusion set is used to drawblood from a suitable large peripheral vein, typically the antecubitalor cephalic vein. Blood is drawn into a 60 cc syringe filled withcitrate anticoagulant at a ratio of approximately 10:1(blood:anticoagulant). After collection of blood, the syringe isinverted several times to ensure mixing with the anticoagulant.

IV. Apparatuses

An apparatus of the present invention or one that is utilized in amethod of the present invention may comprise at least one filtrationcomponent. One or more filters may be utilized in the apparatus or acomponent thereof. A filter may be designed to utilize “cross-flowfiltration” or “cross-flow mode” in which there is recirculation of aretentate across the surface of the membrane filter. In alternativeembodiments, a filter is designed to utilize “dead-end filtration” or“dead-end mode”, which refers to having substantially no tangential flowfiltration.

Microfiltration may be utilized in the invention, which regardsseparation of particles from a fluid. At least one microfiltrationdevice comprises at least one filter having a pore size of about 0.05 μmto about 5 μm. In a specific embodiment of the present invention, cellsand/or cell fragments are separated from at least some fluid, whereinthe microfiltration capabilities of the filter permit passage of thefluid through the pores, thereby retaining the cells and/or cellfragments.

A filtration device may be of any kind, so long as it concentratesnucleated cells, platelets, or both. In preferred embodiments, a devicepreferably separates cells from a portion of the liquid in which theyare suspended, thereby increasing the cellular concentration.

In preferred embodiments, a filtration device may comprise a tubularfilter, as opposed to a disk filter. In additional specific embodiments,the tubular filter is housed in an outer tube comprising hollow tubeswithin it and, in preferred embodiments, the flow through the tubularfiltration device is parallel to the flow through the tubes within thetubular filtration device. In a specific embodiments, the pore size onthe walls of the tubes is about 0.05 μm to about 5 μm, is morepreferably about 0.1 μm to about 1 μm, and is most preferably about 0.2μm to 0.5 μm.

In some embodiments of the present invention, a hollow fiber filtrationdevice is utilized and may be commercially obtained. The devicecomprises, in particular embodiments, a hollow tubular outer covering,wherein inside the hollow tubular outer covering there are individualtubes whose walls are filters; the individual tubes lay in a directionparallel to the length of the tube. In specific embodiments, thedirection of feed of a product through a device is perpendicular to thedirection of flow across the membrane. In some embodiments it generallycomprises tangential flow filtration, which regards the recirculation ofa retentate across the surface of the membrane filter. In alternativeembodiments, there is no tangential flow filtration.

A skilled artisan recognizes that any filtration surface that contactsany of the relevant compositions and/or the concentrate is preferablysterile or readily sterilizable. Commercially available filtration unitscan be sterilized by treating with agents such as gamma irradiation,ethylene oxide, formalin, hydrogen peroxide, or sodium hypochlorite.Sterile filtration units are commercially available for hematologicuses. Syringes and other fluid delivery systems are generallycommercially available in sterile form as are various valves andstopcocks that are designed to attach to syringes and other bloodprocessing products.

An apparatus utilized in methods described herein may be considereddisposable, although they may be reused upon sterilization, such as bygamma sterilization. Filters and syringes may be commercially obtained.In a specific embodiment, the apparatus is plastic. In further specificembodiments, the footprint of the leukoreduction filter is approximately12 cm in diameter and 25 mm thick and/or the footprint of the hollowfiber filter is approximately 120 mm long×20 mm.

V. Addition of Biological Agents to the System

In a preferred embodiment of the present invention a biological agent isincluded in the material delivered to the bone defect (either the cellconcentrate, the scaffold material, or both). Examples includeantibiotics, growth factors, fibrin, bone morphogenetic factors, bonegrowth agents, chemotherapeutics, pain killers, bisphosphonates,strontium salt, fluoride salt, magnesium salt, and sodium salt.

In contrast to administering high doses of antibiotic orally to anorganism, the present invention allows antibiotics to be included withinthe material of the composition for a local administration. This reducesthe amount of antibiotic required for treatment of or prophalaxis for aninfection. Administration of the antibiotic by the material in acomposition would also allow less diffusing of the antibiotic,particularly if the antibiotic is contained within a fibrin matrix.Alternatively, the scaffold material of the present invention may becoated with the antibiotic and/or contained within the scaffold materialor the concentrate, or a combination thereof. Examples of antibioticsare tetracycline hydrochloride, vancomycin, cephalosporins, andaminoglycocides such as tobramycin and gentamicin, and quinoloneantibiotics, such as ciprofloxacin.

Growth factors may be included in the material for a local applicationto encourage bone growth. Examples of growth factors that may beincluded are platelet derived growth factor (PDGF), transforming growthfactor β (TGF-β), insulin-like growth factor-I (IGF-I), insulin-likegrowth factor-II (IGF-II), fibroblast growth factor (FGF),beta-2-microglobulin (BDGF II), bone morphogenetic protein (BMP), growthand differentiation factor-5 (GDF-5), vascular endothelial growth factor(VEGF), or mixtures thereof. The scaffold material of the presentinvention may be coated with a growth factor and/or contained within thescaffold material or the concentrate, or a combination thereof.

Proteins or agents which are accessory to and/or bind to growth factormay be used in the present invention. Examples of the growth factorbinding/accessory protein includes follistatin, osteonectin, sog,chordin, dan, cyr61, thrombospondin, type IIa collagen, endoglin, cp12,nell, crim, acid-1 glycoprotein, and alpha-2HS glycoprotein.

In some embodiments, the compositions of the present invention include acell, such as an osteoblast, endothelial cell, fibroblast, adipocyte,myoblast, mesenchymal stem cell, chondrocyte, multipotent stem cell,pluripotent stem cell and totipotent stem cell, or a musculoskeletalprogenitor cell.

Bone morphogenetic factors may include growth factors whose activity isspecific to osseous tissue including proteins of demineralized bone, orDBM (demineralized bone matrix), and in particular the proteins calledBP (bone protein) or BMP (bone morphogenetic protein), which actuallycontains a plurality of constituents such as osteonectin, osteocalcinand osteogenin. The factors may coat the scaffold material of thepresent invention and/or may be contained within the scaffold material,the concentrate, or a combination thereof.

Angiogenic factors may be included on the scaffold material or in thescaffold material or concentrate, or a combination thereof. Someexamples of angiogenic factors include monobutyrin, dibutyrin,tributyrin, butyric acid, vascular endothelial growth factor (VEGF),erucimide, thymosin Beta 4(TB4), synthetic peptide analogs to heparinbinding proteins, nicotine, nicotinamide, spermine, angiogenic lipids,ascorbic acid and derivatives thereof and thrombin, including analogsand peptide fragments thereof.

Bone growth agents may be included within the scaffold material,concentrate, or both, of the composition of the invention, in a specificembodiment. For instance, nucleic acid sequences that encode an aminoacid sequence, or an amino acid sequence itself may be included in theconcentrate and/or scaffold material of the present invention whereinthe amino acid sequence facilitates bone growth or bone healing. As anexample, leptin is known to inhibit bone formation (Ducy et al., 2000).Any nucleic acid or amino acid sequence that negatively impacts leptin,a leptin ortholog, or a leptin receptor may be included in thecomposition. As a specific example, antisense leptin nucleic acid may betransferred within the composition of the invention to the site of abone deficiency to inhibit leptin amino acid formation, thereby avoidingany inhibitory effects leptin may have on bone regeneration or growth.Another example is a leptin antagonist or leptin receptor antagonist.

The nucleic acid sequence may be delivered within a nucleic acid vectorwherein the vector is contained within a delivery vehicle. An example ofsuch a delivery vehicle is a liposome, a lipid or a cell. In a specificembodiment the nucleic acid is transferred by carrier-assistedlipofection (Subramanian et al., 1999) to facilitate delivery. In thismethod, a cationic peptide is attached to an M9 amino acid sequence andthe cation binds the negatively charged nucleic acid. Then, M9 binds toa nuclear transport protein, such as transportin, and the entireDNA/protein complex can cross a membrane of a cell.

An amino acid sequence may be delivered within a delivery vehicle. Anexample of such a delivery vehicle is a liposome. Delivery of an aminoacid sequence may utilize a protein transduction domain, an examplebeing the HIV virus TAT protein (Schwarze et al., 1999).

In a preferred embodiment the biological agent of the present inventionhas high affinity for a fibrin matrix.

In a specific embodiment, the particle of the present invention maycontain within it or on it a biological agent which would either elutefrom the particle as it degrades or through diffusion.

The biological agent may be a pain killer. Examples of such a painkiller are lidocaine hydrochloride, bipivacaine hydrochloride, andnon-steroidal anti-inflammatory drugs such as ketorolac tromethamine.

Other biological agents that may be included in the suspension materialor contained on or in the concentrate and/or scaffold material of thepresent invention are chemotherapeutics such as cis-platinum,ifosfamide, methotrexate and doxorubicin hydrochloride. A skilledartisan is aware which chemotherapeutics would be suitable for a bonemalignancy.

Another biological agent which may be included in the concentrate and/orscaffold material is a bisphosphonate. Examples of bisphosphonates arealendronate, clodronate, etidronate, ibandronate,(3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD), zoledronate,dichloromethylene bisphosphonate, aminobisphosphonatezolendronate andpamidronate.

The biological agent may be either in purified form, partially purifiedform, commercially available or in a preferred embodiment arerecombinant in form. It is preferred to have the agent free ofimpurities or contaminants.

VI. Combined Therapy

A skilled artisan recognizes that the present invention provided hereinin some embodiments is used in conjunction with another therapy for theindividual. For example, in the embodiments wherein a bone defect isbeing treated with methods and/or compositions of the present invention,the patient may also receive standard bone therapy treatments. Standardbone therapy treatments for repair of bone defect include surgery,additional bone grafting, antibiotic administration, implant use,external stimulation such as low-intensity ultrasound or electromagneticpulses, and so forth.

VII. Pharmeceuticl Compositions and Routes of Administration

Compositions of the present invention will have an effective amount ofan osteogenic material for therapeutic administration. In someembodiments, the compositions are used in combination with an effectiveamount of a second compound (second agent) that is a bone therapy agentas exemplified above. Such compositions will generally be dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.The term “effective” as used herein refers to providing bone growthproperties, preventing degeneration, strengthening of bone, preventingfracture, healing fracture, and so forth.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, orhuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredients,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients, such as other bone therapy agents, can also beincorporated into the compositions.

The compositions of the present invention are advantageouslyadministered in a form suitable for application to a bone defect. Thecomposition form may be injectable compositions, either as liquidsolutions or suspensions, although solid forms suitable for solution in,or suspension in, liquid prior to injection also may be prepared. Inother embodiments, the composition is applied in a non-injectablemanner. Examples of such include applying a cell concentrate alone or ina mixture with a scaffold material using a utensil, such as a scoop,scoopula, spatula, rod, spoon, syringe, pipette, forceps, measuredspoon, or any such medically approved delivery vehicle.

In particular embodiments of the present invention, a solutioncomprising the desired cell concentrate comprises phosphate buffer,dextrose, salt, dextran 40, dextran 70, or a mixture or combinationthereof. Other pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters, suchas theyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Preservatives include antimicrobialagents, anti-oxidants, chelating agents and inert gases, although inspecific embodiments the concentrate is used soon after concentration atthe point-of-care. The pH and exact concentration of the variouscomponents in the pharmaceutical are adjusted according to well-knownparameters.

All of the essential materials and reagents required for bone therapymay be assembled together in a kit. When the components of the kit areprovided in one or more liquid solutions, the liquid solution preferablyis an aqueous solution, with a sterile aqueous solution beingparticularly preferred. In the kit, there may be an apparatus to collecta physiological solution, at least one filter and/or filter apparatus, ascaffold material, a utensil and/or platform to combine the cellconcentrate and scaffold material, and/or a combination thereof.

The kits of the present invention also will typically include a meansfor containing the vials in close confinement for commercial sale suchas, e.g., injection or blow-molded plastic containers into which thedesired vials are retained. Irrespective of the number or type ofcontainers, the kits of the invention also may comprise, or be packagedwith, an instrument for assisting with the injection/administration orplacement of the ultimate complex composition within the body of ananimal. Such an instrument may be a scoop, scoopula, spatula, rod,spoon, syringe, pipette, forceps, measured spoon, or any such medicallyapproved delivery vehicle.

The concentrate or concentrate/scaffold material can also be comprisedwith a solvent or dispersion medium comprising, for example, blood,plasma, water, sugar, salt, or suitable mixtures thereof. The preventionof the action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

VIII. Kits

In some embodiments of the present invention, kits are provided, such asto perform methods described herein and/or generate compositionsdescribed herein. In a particular embodiment, these kits are utilizedfor treatment of a bone defect. In some particular embodiments, there isa kit for preparing a cell concentrate, comprising a first filtrationdevice and a second filtration device, both of which are housed in asuitable container. The first filtration device may be a nucleated cellfiltration device and/or the second filtration device is amicrofiltration device, such as, for example, a hollow fiber filtrationdevice.

The kit may further comprise a scaffold material housed in a suitablecontainer. The kit may further comprise a biological agent housed in asuitable container. An exemplary biological agent includes a growthfactor, an antibiotic, a strontium salt, a fluoride salt, a magnesiumsalt, a sodium salt, a bone morphogenetic factor, an angiogenic factor,a chemotherapeutic agent, a pain killer, a bisphosphonate, a growthfactor binding/accessory protein, a cell, a bone growth agent, or amixture thereof. In a specific embodiment, the growth factor is selectedfrom the group consisting of platelet derived growth factor (PDGF),transforming growth factor β (TGF-β), insulin-related growth factor-I(IGF-I), insulin-related growth factor-II (IGF-II), fibroblast growthfactor (FGF), beta-2-microglobulin (BDGF II), nerve growth factor (NGF),epidermal growth factor (EGF), keratinocyte growth factor (KGF), bonemorphogenetic protein (BMP) and a mixture thereof. In a further specificembodiment, the antibiotic is selected from the group consisting oftetracycline hydrochloride, vancomycin, cephalosporins, quinolone,aminoglycocides, and a mixture thereof. In a specific embodiment, thequinolone is ciprofloxacin. In a specific embodiment, the aminoglycocideis tobramycin or gentamicin.

In other embodiments, there is a kit for treating a bone defect,comprising a plurality of cells housed in a suitable container, whereinthe cells are nucleated cells, platelets, or both. Such a kit maycomprise a scaffold material housed in a suitable container and/or abiological agent housed in a suitable container.

A kit may comprise an apparatus for the preparation of a cellconcentrate obtained by a method or methods described herein. A kit ofthe present invention may comprise, at least, any apparatus suitable toperform any method described herein.

The kits provided herein may be considered single use disposable kits.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Dual Filtration of Physiological Solution

An in vitro study is performed to determine the efficacy of the filterdevice in concentrating nucleated cells, such as mesenchymal stem cells,and platelets within bone marrow aspirate, although in some embodimentsblood or a mixture of blood and bone marrow may be utilized. A volume ofthe exemplary marrow aspirate, such as 25 mL, is procured from an animaldonor(s) (such as, for example, sheep). Each sample is divided, whereinhalf the sample is not processed and half the sample is processed by thefiltration device(s) described herein. The volume of the unprocessedsample (V0) and that of the sample after it has been processed (V1) ismeasured. Nucleated cell count, mesenchymal stem cell count, andplatelet count is determined by hemocytometry and flow cytometry foreach unprocessed and processed sample. Additionally, in order toquantify the concentration of mesenchymal stem cells that differentiatetowards an osteoblastic lineage, cells from the unprocessed marrow andthe cell concentrate are cultured in osteogenic media and assayed forosteoblastic markers, such as, for example, alkaline phosphatase.Nucleated cell count, platelet count, and alkaline phosphatase activityis normalized to sample volume (V0 or V1) to yield a measure ofnucleated cell, mesenchymal stem cell, and platelet concentration. Thefiltration of the marrow aspirate should result in a significantretention of mesenchymal stem cells and platelets (preferably greaterthan about 60%) and a significant reduction in volume (preferablygreater than about 80%) compared to unprocessed marrow. This shouldresult in an approximately 3-fold or greater increase in cellconcentration compared to unprocessed marrow aspirate.

In light of the demonstration of the efficacy of the filter device inconcentrating marrow in vitro, an implantation study is performed todetermine acceleration of bone healing by the concentrated marrowsuspension in an animal model (such as, for example, sheep). Autologousmarrow aspirate is processed through the filter instrument to increasethe concentration of mesenchymal stem cells and platelets. Theconcentrated marrow suspension is combined with an osteoconductivescaffold material and, if necessary, a carrier material, and implantedin a critical-sized defect (such as, for example, a defect that will notspontaneously heal when left empty). The concentrated marrowaspirate+scaffold group is compared to an unprocessed marrowaspirate+scaffold group in terms of bone healing. Bone healing ismeasured radiographically, mechanically, and/or histologically bystandard means in the art. The concentrated marrow aspirate grouppreferably shows superior bone healing compared to the unprocessedmarrow aspirate group.

Example 2 Description of Exemplary Filter Configurations

Exemplary filter configurations in the present invention may be asfollows:

1. Leukofilter only (see FIG. 4);

2. Leukofilter+Hollow Fiber Filter (Cross-flow mode) (see FIGS. 4, 5,6);

3. Leukofilter+Hollow Fiber Filter (Dead end mode) (see FIGS. 4, 7, 8);and/or

4. Use of a Fat Reduction Filter with any of the above threeconfigurations (FIG. 9).

In FIGS. 4A-4D, there is a schematic showing operation of a sole (orfirst, as in some embodiments described below) filter (such as aleukocyte reduction-type filter), to selectively recover osteogeniccells (i.e. platelets and nucleated cells) from a physiologicalsolution. In the Fill step (FIG. 4A), a physiological solutioncomprising both osteogenic and non-osteogenic cells and optionallycomprising anticoagulant is injected into a collection bag 10. In afiltration step (FIG. 4B), the physiological solution passes from thecollection bag 10 through the leukocyte reduction-type filter 12, suchas by gravity feed. After passing through the filter where cells such asnucleated cells, platelets, or a mixture thereof are retained, theremainder of the physiological solution and its constituents (such asred blood cells) flow into the drain bag 14. In a back-flush step (FIG.4C), valves 11 and 13 to the collection bag 10 and drain bag 14,respectively, are closed. The valves 15 and 17 to the syringes 9 and 19,respectively, are opened. In the recovery step (FIG. 4D), osteogeniccells are backflushed from the filter 12 when recovery solution fromsyringe 19 flows through valve 17 to force the osteogenic cells intosyringe 9. The collection in syringe 9 comprises the recovered cells andin some embodiments is not processed further but is applied to a bonedefect, optionally after combining the cells with a scaffold material.In alternative embodiments, the cells in syringe 9 are referred to as afeed for a subsequent step in the process, and the syringe may bereferred to as a feed syringe 9.

FIG. 5 shows a schematic drawing of an operation of a second filter,such as a hollow fiber filter, to concentrate the osteogenic cells(i.e., platelets and nucleated cells) recovered from the first filter 12in FIG. 4, for example. A syringe comprising the osteogenic cells, suchas the exemplary syringe 9 from FIG. 4, is connected to the hollow-fiberfilter 30, which is operated in cross-flow mode. In cross-flowfiltration, the feed stream is recirculated between a feed syringe 9,for example, and retentate syringe 36 tangentially to the membrane inthe hollow-fiber filter 30, establishing a pressure differential acrossthe membrane. This causes some of the particles to pass through themembrane. Remaining particles continue to flow across the membrane.Using an appropriate membrane pore size (such as about 0.2 to 0.5 μm),recovery solution, but not cells, are able to pass through the filtermembrane of the hollow fiber filter 30. FIG. 6 demonstrates a schematicfor showing concentrate osteogenic cell suspension resulting fromoperation of a second filter. Cross-flow filtration fractionates aportion of the recovery solution from the suspended cells, therebyincreasing the concentration of the cells in the retentate syringe 36.

FIG. 7 provides a schematic showing operation of a second filter, suchas a hollow fiber filter 30, to concentrate osteogenic cells (i.e.,platelets and nucleated cells) recovered from a first filter. Theexemplary syringe, such as syringe 9 from FIG. 4, containing theosteogenic cells is connected to the hollow fiber filter 30, which isoperated in dead-end mode (this is accomplished by capping the retentatesyringe port 34). Unlike cross-flow filtration, dead end filtration doesnot involve the recirculation of the feed stream between an exemplaryfeed syringe 9 and retentate syringe tangentially to the membrane.Pressure from the advancing feed syringe establishes a pressuredifferential across the membrane. This causes some of the particles topass through the membrane. Using an appropriate membrane pore size (suchas about 0.2 to 0.5 μm), recovery solution, but not cells, are able topass through the filter membrane to the hollow fiber filter 30. FIG. 8is an exemplary schematic showing a concentrate osteogenic cellsuspension resulting from operation of a second filter in dead end mode.Dead end filtration fractionates a portion of the recovery solution fromthe suspended cells, thereby increasing the concentration of the cellsremaining in the feed syringe 9.

Example 3 Description of Cell Concentrate Characteristics

In a particular embodiment of the present invention, the final cellconcentrate must have at least one of the following characteristics: 1)nucleated cells per unit volume greater than in the startingphysiological solution; 2) platelets per unit volume greater than in thestarting physiological solution; and/or 3) upon activation of platelets,the concentration of growth factors is greater than that of anequivalent volume of the starting physiological solution. Examples ofgrowth factors are platelet-derived growth factor (PDGF), transforminggrowth factor beta (TGF-β), vascular endothelial growth factor (VEGF),epithelial growth factor (EGF), and insulin-like growth factor (IGF).

In particular embodiments, the method to obtain the cell concentrateand/or the cell concentrate itself may have the following attributes: 1)leukocyte concentration of at least about six times that of baselineblood value; 2) substantially no exogenous animal-derived orhuman-derived constituents in backflush solution (although inalternative embodiments an exogenous animal-derived or human-derivedconstituent is added, such as the exemplary human serum albumin); 3)substantially no exogenous animal-derived or human-derived constituentsin clotting initiator; and/or 4) filter should activate less than about25% of platelets (Babbush et al., 2003).

In other embodiments, the method to obtain the cell concentrate and/orthe cell concentrate itself more preferably has the followingattributes: 1) a sole (or first) filter processes a minimum of about 60mL and maximum of about 120 mL of blood; 2) blood is mixed with at leastone anticoagulant; 3) recovery solution comprises biocompatible and/ornon-pyrogenic constituents; 4) filtration step(s) takes about 10 minutesor less; 5) filter recovers at least about 70% of platelets from blood;6) filter concentrates platelets at least about 6-fold compared tobaseline physiological solution (such as blood or bone marrowconcentrate) value; 7) platelet concentrate comprises sufficientclotting factors for clot initiation (exemplary clotting factors includefibrinogen, Factor V, Factor VII, Factor VIII, Factor IX, Factor X,Factor XI, Factor XII, Factor XIII, prekallikrein, prothrombin, tissuefactor, von Willebrand Factor (vWF) (proteins may be monitored duringthe methods, such as fibrinogen concentration being measured by ELISA));8) platelet concentrate clots within about 5 minutes after addition ofclot initiator; 9) final clot volume comprises about 5 to 10 mL; and/or10) once clotted, the concentration of growth factors (such as, forexample, PDGF, TGF-β, VEGF, EGF, and IGF) comprises at least about 6times that of an equivalent or substantially equivalent volume ofclotted blood.

In specific embodiments of the present invention, materials utilized inthe present invention meet governmental regulatory standards in the art,such as being FDA-approved.

Example 4 Methods to Measure Cell Concentrate Characteristics

Nucleated Cell (Leukocytes) and Platelet Counts (from Wintrobe'sClinical Hematology, 10th ed.)

By exemplary means, leukocytes and platelets can be enumerated by eithermanual methods and/or automated hematology analyzers, both of which arewell known in the art. Manual counts are carried out after appropriatedilution of the sample in a hemocytometer, a specially constructedcounting chamber that contains a specific volume. Cells may then becounted, for example, with a microscope.

Automated hematology analyzers increase the accuracy and speed ofanalysis, and, thus, may be preferable to manual counting. Two exemplarymajor types of automated counters may be used to enumerate leukocytesand platelets: aperture-impedance counters and optical method counters.Aperture-impedance counters include the Coulter (Hialeah, Fla.), theSysmex (Baxter Diagnostics, Waukengan, Ill.), and some Cell-Dyneinstruments (Abbott Diagnostics, Santa Clara, Calif.), for example.Optical method counters include Technicon (Bayer Diagnostics, Kent,Wash.) and some Cell-Dyn instruments, for example.

Cell counts are determined for the original physiological solution andthe final post-processed cell concentrate.

Growth Factor Quantification

Enzyme-linked immunosorbent assays (ELISA) are used to quantify growthfactor concentrationk, in exemplary embodiments. Growth factorconcentrations are determined for the original physiological solutionand the final post-processed cell concentrate after platelet activation.An appropriate platelet activator, such as thrombin, a calcium chloridesolution, adenosine diphosphate (ADP), or a combination thereof, may beused to activate platelets. After sufficient time to allow plateletactivation and growth factor release (such as less than about one hour),growth factor concentration is quantified, such as by using commerciallyavailable ELISA kits.

Example 5 Methods to Remove Fat from the Physiological Solution

In some embodiments of the present invention an additional step ormeans, such as a filter, is incorporated into the design in order toremove fat from a physiological solution, such as particles (which mayinclude adipocytes). The fat may be cellular or non-cellular in form.Thus, a skilled artisan recognizes that this refers to adipocytes,congealed fat that is non-cellular in nature, or both. A physiologicalsolution is passed through this step or means, such as an exemplaryfilter referred to herein as a fat reduction filter, in order tosubstantially reduce (or in some embodiments eliminate) a quantity offat. The removal of fat will result in faster flow and better osteogeniccell capture as the physiological fluid passes through a subsequentfilter(s) in the process. In preferred embodiments, enough fat isremoved so that there is no occlusion, blocking, clogging and such ofthe filter(s). A skilled artisan recognizes that complete removal of fator adipocytes is desirable, but unnecessary so long as filtration isachievable.

Commercially available filters that have been used clinically to removemicroaggregates from blood may be suitable for this application.Examples include but are not limited to standard blood transfusionfilters, such as the SQ40SJKL (Pall Corp., Port Washington, N.Y.) andLipiGuard (Pall Corp., Port Washington, N.Y.).

FIGS. 9A-9E show a schematic of the incorporation of a fat reductionfilter 8 into the device used to prepare an osteogenic cell concentrate.The physiological solution is passed via syringe 6 through the exemplaryfat reduction filter 8 component into a collection bag 10 (FIG. 9A). Thefluid, reduced in fat particle content, is then processed through theleukofilter 12 as shown in FIGS. 4 and 9. If necessary, the cellsuspension recovered from the leukofilter 12 is then processed throughthe hollow fiber filter 30 in either cross-flow mode (FIGS. 5 and 6) ordead-end mode (FIGS. 7 and 8).

Example 6 Bone Marrow Aspirate (BMA) Filtration in Immature New ZealandWhite Rabbits

BMA was taken from two skeletally immature New Zealand White rabbits andprocessed through an exemplary filter of the present invention todemonstrate an increase in the concentration of nucleated cells (a cellpopulation that, in some embodiments, includes undifferentiated cells,such as osteoprogenitors). This study showed that the filters were ableto concentrate BMA from immature New Zealand White rabbits within atargeted range (about 6 to 10-fold) in a specified timeframe (underabout 15 minutes).

Materials and Methods

Blood and bone marrow aspirate (BMA) were drawn from three skeletallyimmature New Zealand White rabbits. Each rabbit was 2 months old andweighed approximately 1.4 kg at the time of the procedure. The rabbitswere not treated with any medications or steroids prior to BMA harvest.Anesthesia was induced by intramuscular administration of 50 mg/kg ofketamine and 10 mg/kg of Xylazine. Approximately 2-mL of blood was drawnfrom the aorta of each rabbit and mixed with 0.3-mL of heparinizedsaline. The volume of blood drawn from each rabbit is listed in Table I.Bone marrow was bilaterally aspirated from the proximal tibias of eachrabbit. A 0.5 to 1-cm incision was made on the medial aspect of anteriorproximal metaphysis of the tibia. An 18-g needle was rotated gently toperforate the cortical bone and enter the marrow cavity. If the initialneedle clogged during the perforation, the needle was replaced with anew one. Negative pressure was established by drawing the plunger backuntil marrow began flowing into the syringe; the pressure was thenreduced, and the marrow was collected for about 10 seconds. For eachtibia, two to 3.5-mL of BMA was aspirated using an 18-g needle into a 10cc disposable syringe filled with 0.5-mL of heparinized saline (1000units/mL). The syringe was detached and was inverted several times toensure complete mixing, and then the BMA was transferred to a sterile,graduated plastic test tube. The volume of BMA drawn from each rabbit islisted in Table II.

The initial count of nucleated cells in each sample was determined bylysing the red blood cells in a 2% acetic acid solution. One hundred μLof aspirate was mixed with 900-μL of acetic acid. A portion of theresultant cell suspension was injected into a hemocytometer grid andcells were counted using a compound microscope (BX60, Olympus OpticalCo., Ltd., Japan) at 200× magnification. Nucleated cell concentrationwas calculated as:N=C×SF×D _(acetic acid)

where N=nucleated cell concentration; SF scale factor of hemocytometergrid=104; and D_(acetic acid)=dilution factor in acetic acid=10. Atleast two replicate counts were performed for each sample.

After determination of initial nucleated cell count, four-ml of BMA fromeach rabbit were pooled to yield a combined volume of 12-mL. The pooledBMA sample was then passed through the exemplary filtration device asshown schematically in FIGS. 5, 6, and 10. In FIGS. 10A-10E, theaggregate filter 7 may remove large particles of material, which incertain embodiments comprises fat particles.

This particular embodiment comprises a system having at least a one-stepprocess utilizing a leukoreduction filter to capture osteogenic cellsand, optionally, a hollow fiber filter to concentrate the capturedcells. The leukoreduction filter (P/N B-1462G; L/N 312700) and recoverysolution (L/N SLS051303) were manufactured by Pall Medical, Inc. (PortWashington, N.Y.). The hollow fiber filter (P/N X12E-100-20N; L/NDH157/N) was made by Spectrum Laboratories, Inc. (Rancho Dominguez,Calif.). The nucleated cell concentration of the ACS-processed cellsuspension was determined as described in the preceding paragraph.

Results

The mean nucleated cell counts of the blood, pre-processed BMA, andpost-processed BMA were 6.0×10⁶, 20.1×10⁶, and 131×10⁶ cells/ml (TablesI, II, and III, respectively). Representative fields of view of cells inthe hemocytometer are shown in FIG. 11. The nucleated cell count of thepre-processed BMA was approximately 3.5 times that of blood (Holdrinetet al., 1980). This indicates that bone marrow cells were collected inthe aspirate; BMA composed entirely of peripheral blood would have anucleated cell count similar to that of blood. Twelve mL of BMA pooledfrom the three donor rabbits was processed through the ACS prototype inapproximately 5 minutes. The final volume was 0.6-mL, representing a20-fold decrease in volume. The cell concentration of the post-filteredsample was approximately 6.5 times that of the pre-processed BMA. Giventhat a 20-fold decrease in volume resulted in a 6.5-fold increase incell concentration, the cell recovery efficiency of the filtrationprototype was 33%.

This study showed that the prototype filters were able to concentrateBMA from skeletally immature New Zealand White rabbits within thetargeted range (about 6- to 10-fold) and timeframe (under about 15minutes). Given that age-related changes in marrow cellularity and fatcomposition in rabbits are known (Kita et al., 1987; Bigelow andTavassoli, 1984), in alternative embodiments the present inventionprovides filtration of concentrate nucleated cells in BMA fromskeletally mature rabbits.

TABLE I Volume and nucleated cell counts for blood samples fromindividual rabbits. Blood Volume Replicate Cell Nucleated Cell Sample(ml) Counts Concentration (10⁶/ml) Rabbit 1 2.8 88; 119; 91 9.7 Rabbit 21.4 32; 48; 40 4.0 Rabbit 3 1.8 43; 49; 36 4.2 Mean = 6.0 ± 3.2

TABLE II Volume and nucleated cell counts for unprocessed bone marrowaspirate (BMA) samples from individual rabbits. BMA Volume ReplicateCell Nucleated Cell Sample (ml) Counts Concentration (10⁶/ml) Rabbit 1 8214; 229 22.2 Rabbit 2 7 203; 180 19.2 Rabbit 3 5 172; 213; 183 18.9Mean = 20.1 ± 1.8

TABLE III Volume and nucleated cell counts for filtration-processed bonemarrow aspirate. Four ml of marrow was taken from each of the threerabbits and processed through the filtration system. Starting FinalNucleated Cell Volume Volume Replicate Concentration Sample (ml) (ml)Cell Counts (10⁶/ml) Filtration- 12 0.6 989; 1590; 131 ± 30 ProcessedBMA 1335

Example 7 Method to Activate Platelets and Coagulate Cell Concentrate

A platelet activation agent (e.g., calcium chloride solution orthrombin, or a combination thereof) may be added to the cell concentratein order to activate platelets and induce coagulation. The addition ofthe platelet activator to the cell concentrate will result in a highergrowth factor concentration (due to increased platelet activation) andbetter handling characteristics (due to coagulation), in preferredembodiments.

The platelet activator should be added to the cell concentrate at anappropriate ratio to induce rapid platelet activation (less than 30minutes) and coagulation. For example, a 1:10 activator:cell concentrateratio is used when the activator is a CaCl₂/thrombin (100 units/ml)solution.

A portion of the cell concentrate prepared for each blood donor is addedto a glass beaker, and a coagulum is formed by the addition of anappropriate platelet activator. The mixture is allowed to undergo clotretraction for an appropriate time period (at least about 30 minutes).The contents of the beaker will then be transferred to centrifuge tubesand centrifuged at about 1,000 g for about 30 minutes. The growth factorconcentration in the supernatant is quantified by commercially availableenzyme-linked immunosorbent assays (ELISA) kits, in exemplaryembodiments. For reference, this experiment is repeated for: (i) anunprocessed blood sample from each donor; and/or (ii) a portion of thecell concentrate to which no platelet activator is added.

Example 8 Preparation of a Platelet Rich Concentrate (PRC) from HumanBlood Using a Single Filter

Whole blood was taken from healthy human donors and processed through anexemplary filter of the present invention to demonstrate at least anincrease in the concentration of platelets and platelet-derived growthfactors. This study showed that processing of human blood through asingle filter (for example, a leukoreduction filter) was able toconcentrate platelets and growth factors above baseline values observedfor unprocessed blood.

A schematic of the filter that was evaluated is shown in FIG. 12. Eachprototype comprised two standard 150-cc blood collection bags, aleukoreduction filter, and two ports for syringe attachment. To evaluatethe filters, units of whole blood (each unit was 450-ml) were collectedfrom healthy human volunteers into blood bags containing 50-ml ofAnticoagulant Citrate Dextrose, formula A (ACD-A). Sixty ml aliquotswere transferred into a 60-ml syringe. The contents of the syringe werethen injected into a blood bag comprising 10-ml of platelet capturesolution A (water for injection). In some cases, 120-ml ofanticoagulated blood was injected into a bag comprising 20-ml ofsolution A to determine if the filters could effectively process adouble dose of blood. The blood bag comprising the diluted blood samplewas attached to a platelet recovery filter (Purecell PL, Pall Medical,Inc., Port Washington, N.Y.). The filtration height (vertical distancebetween top of blood line in collection bag and entry point into drainbag) was adjusted to 12.5 inches. The diluted blood sample was thenfiltered at room temperature. The filtration time for each sample wasrecorded. After filtration was complete, the filter was backflushed witha syringe filled with 7-ml of platelet recovery solution B (5% salinesolution) and 13-cc of air. The contents of the filter were backflushedinto a 20 cc syringe. The recovered cellular suspension was transferredfrom the syringe into a 15-ml centrifuge tube. The total volume of therecovered platelets was measured using the graduated markings on thecentrifuge tube.

For each donor, platelet concentration was measured for the recoveredcell suspension and whole blood samples using a Cell Dyn HematologyAnalyzer (Abbott Labs, IL). ELISAs for PDGF-AB, TGF-β1, and VEGF-A(Quantikine, R&D Systems, Minneapolis, Minn.) were performed on therecovered suspension, the recovered suspension+10% CaCl₂/thrombin (5000Units/ml), and whole blood. Significant differences in means weredetermined by ANOVA at a 95% confidence level.

Filtration time ranged from 10 to 15 minutes for the 60-ml blood samples(n=20) and from 45 to 90 minutes for the 120-ml samples (n=9). The flowrate of the 120-ml samples was slowed by backpressure from the drain bagas it filled to near maximum capacity. The final volume of the recoveredcell suspension (for both starting blood volumes) ranged from 9 to10-ml, averaging 9.5-ml.

The mean platelet recovery was approximately 65% for both the 60-ml and120-ml blood samples and did not differ significantly between the twogroups (FIG. 13). The mean platelet concentration factors were 4.1±0.4fold and 8.3±1.2 fold for the 60-ml and 120-ml samples, respectively,compared to whole blood (p<0.05).

The mean concentrations of PDGF-AB, TGF-β1, and VEGF-A in whole bloodand the platelet concentrate are shown in FIGS. 14-16, respectively(n=15). The cell concentrate produced by the filter showed 16-fold,25-fold, and 14-fold increases in PDGF, TGF-β, and VEGF concentrations,respectively, compared to whole blood (p<0.05 in all cases). When thecell concentrate was activated with calcium chloride/thrombin, theconcentration factor increased to 26-fold and 34-fold for PDGF andTGF-β, respectively (p<0.05 in both cases). The VEGF concentrationfactor dropped from 14-fold to 3-fold upon activation with calciumchloride/thrombin. In a specific embodiment, this is related to theapparent ability of fibrin to bind VEGF, possibly masking the ELISAepitope.

This study showed that an exemplary filter of the present invention isuseful for conveniently preparing a platelet rich concentrate from wholeblood. The filtration process took 10 to 15 minutes and produced a final9.5-ml product with greater than 4-fold enrichment in platelets.Additionally, the concentrations of PDGF-AB, TGF-β1, and VEGF-A weresignificantly higher in the platelet concentrate compared to wholeblood.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

Patents

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PUBLICATIONS

-   Babbush C A, Kevy S V, Jacobson M S. An in vitro and in vivo    evaluation of autologous platelet concentrate in oral    reconstruction. Implant Dent. 2003; 12(1):24-34.-   Bigelow C L and Tavassoli M, “Fatty involution of bone marrow in    rabbits”, Acta Anat (Basel), 118(1):60-4 (1984).-   Connolly, J., Guse, R., Lippiello, L., Dehne, R. Development of an    Osteogenic Bone-Marrow Preparation, J. Bone and Joint Surgery, vol.    71-A, no. 5, pp. 684-691, 1989.-   Holdrinet R S G, Egmond J V, Wessels J M C, and Haanen C, “A method    for quantification of peripheral blood admixture in bone marrow    aspirates”, Exp. Hemat., 8(1):103-7 (1980).-   Kita K, Kawai K, and Hirohata K, “Changes in bone marrow blood flow    with aging”, J Orthop Res, 5(4):569-75 (1987).-   Takigami, H., Muschler, G. F., et al. Spine Fusion using Allograft    bone matrix enriched in bone marrow cells and connective tissue    progenitors, Trans. of 48^(th) Orthopaedic Res. Soc., p. 807, 2002.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1. A method of treating a bone defect in an individual comprising thesteps of: obtaining a physiological fluid comprising nucleated cells,platelets, plasma and red blood cells, which has not been previouslysubjected to centrifugation, preparing a cell concentrate from thephysiological fluid comprising combining a hypotonic fluid with thephysiological fluid, the hypotonic fluid being in an amount which doesnot cause the cells to lyse in the physiological fluid/hypotonic fluidcombination, subjecting the combined physiological fluid/hypotonic fluidto a first filtration device to produce a filter retentate, which is afirst product, and a permeate fluid, wherein said filter retentatecomprises platelets and nucleated cells per unit volume greater than inthe physiological fluid, and wherein said permeate fluid comprisesplasma and red blood cells, removing the filter retentate from thefilter, and subjecting the filter retentate to a second filtrationdevice to produce a cell concentrate, which is a second product,delivering the second product to the bone defect in the individual. 2.The method of claim 1, wherein the bone defect comprises a break,fracture, void, diseased bone, loss of bone, brittle bone, weak bone,bone injury, or bone degeneration.
 3. The method of claim 1, wherein themethod further comprises the step of admixing a scaffold material withsaid second product.
 4. The method of claim 3, said method furthercomprising the step of admixing a biological agent with the scaffoldmaterial.
 5. The method of claim 4, wherein the biological agent is inthe scaffold material or is on the scaffold material or both.
 6. Themethod of claim 1, wherein said physiological fluid is blood, bonemarrow aspirate, or a mixture thereof.
 7. The method of claim 1, whereinsaid physiological fluid is obtained from said individual.
 8. The methodof claim 1, wherein said method occurs in a hospital facility or ahealth care provider facility.
 9. The method of claim 1, wherein themethod further comprises administering to the individual an additionalbone defect therapy.
 10. The method of claim 9, wherein said additionalbone defect therapy comprises fracture repair, surgery, bone excision,implant delivery, external stimulation, or a combination thereof. 11.The method of claim 1, wherein said nucleated cells comprise stem cells,connective tissue progenitor cells, osteoprogenitor cells,chondroprogenitor cells, or a mixture thereof.
 12. The method of claim11, wherein said stem cells are mesenchymal stem cells, hematopoieticstem cells, or a mixture thereof.
 13. The method of claim 1, whereinsaid delivering the second product to the individual comprises applyingthe second concentrated product directly to the bone defect.
 14. Themethod of claim 13, wherein the applying is with a scoop, scoopula,syringe, rod, tube, or spatula.
 15. The method of claim 1 said methodfurther comprising the step of admixing a biological agent with thesecond product.
 16. The method of claim 1, wherein said method furthercomprises the step of subjecting the physiological fluid to a fatreduction step.
 17. The method of claim 16, wherein the fat reductionstep comprises subjecting said physiological fluid to a fat reductionfiltration.
 18. The method of claim 1, wherein the hypotonic solutioncomprises sodium chloride.